Gene Therapy

1. GENE THERAPY
(DNB Solved answers, Dr.Padmesh.V)
*DEFINITION OF GENE THERAPY:
“ Novel approach to treat,cure or ultimately prevent a disease by changing the expression of a person’s
genes”
*STEPS IN GENE THERAPY:
1. Identification of the defective gene.
2. Cloning of normal healthy gene.
3. Identification of target cell / tissue / organ.
4. Insertion of the normal functional gene into the host DNA.
Method: Introduction of FUNCTIONAL GENES into appropriate cells
Transferred gene (TRANSGENE) encodes & produces proteins
The Proteins encoded by Transgene corrects the disorder.
*GENE THERAPY APPROACHES Two ways to deliver genes:
1. Ex vivo approach 2. In vivo approach
1. Ex vivo approach:
-Target cells are removed from the body and grown in vitro.
-The gene is then introduced into the cultured cells.
-These cells are then re-introduced into the same individual
-Examples: Fibroblast cells, Hematopoietic cells.
2. In vivo approach: (Direct Gene Transfer)
-Cloned therapeutic gene is introduced directly into the affected tissue, without removing cells from
the body.
-Specially designed vehicles are needed.
-Examples are: Used in cells of Lungs, Brain

2. *METHODS OF GENE DELIVERY:
1. PHYSICAL METHODS:
-Parenteral injection -Microinjection -Aerosol -Gene gun
2. CHEMICAL METHODS:
-Calcium phosphate -DEAE-Dextran -Liposomes
3. BIOLOGICAL METHODS:
Viral Vectors like: -Retrovirus -Adenovirus -HSV
4. NEO-ORGAN IMPLANTS
5. TISSUE TRANSPLANTATION
6. HUMAN ARTIFICIAL CHROMOSOMES
7. OTHERS:
-Receptor mediated delivery -Virally directed enzyme prodrug therapy
OF THESE, THE COMMON VECTORS USED FOR GENE THERAPY:
1.Retro viruses 2. Adeno viruses 3. Liposomes
1. RETRO VIRUSES:
-Retroviruses used in gene therapy are made incapable of independent replication,to prevent side
effects associated with infectivity.
-Retroviruses are used ONLY in EX VIVO THERAPY.
Advantages:
-Chromosomal integration & stable modification of target cells.
Disadvantages:
-Uncontrolled integration; May be oncogenic.
-Cannot infect non-dividing cells.

3. 2. ADENO VIRUSES:
-Second most commonly used delivery system in gene therapy.
-Adenoviruses can be produced at high titres in cultures.
Advantages:
-Can infect non-dividing cells,thus suitable for gene therapy of Cystic fibrosis, DMD.
-Non-integration to chromosome. Avoids the risks of uncontrolled integration.
-Efficient gene transfer.
Disadvantages:
-Transient expression of gene due to episomal integration.
-Provokes immune response.
3.LIPOSOMES:
-These are lipid bilayers surrounding an aqueous vesicle.
-Can be used to introduce foreign DNA into a target cell.
Advantages:
-Safer when compared to Viral vectors.
-Can carry large DNA molecules.
Disadvantages:
-Inefficient transfer.
-Transient expression.
*TYPES OF GENE THERAPY: 1. SOMATIC CELL THERAPY 2. GERM LINE THERAPY
1.SOMATIC CELL THERAPY:
-Insertion of therapeutic gene into somatic cells like fibroblasts,myoblasts,epithelial cells, nervous
cells,glial cells etc.
-This can correct the genetic defect in the patient
-However,in somatic cell therapy, Transgene cannot be passed on to the siblings etc.

4. 2. GERMLINE THERAPY:
-Introduction of the foreign gene into germ cells like sperm / ovum / fertilized egg.
-Results in expression of modified features in both somatic as well as germ cells of the offspring.
-Considered unethical,and is not advocated in humans.
*VARIOUS STRATEGIES FOR GENE THERAPY:
1. GENE AUGMENTATION THERAPY
2. TARGETED MUTATION CORRECTION
3. INHIBITION OF GENE EXPRESSION
4. GENE THERAPY TO ACHIEVE PHARMACOLOGICAL EFFECTS
1. GENE AUGMENTATION THERAPY:
-If a disease is caused by a mutation causing loss of function, introduction of a FUNCTIONAL COPY OF
THE GENE into the cell will restore the normal function of the gene.
Examples: 1.Deficiency of ADA 2. Haemophilia
2. TARGETED MUTATION CORRECTION:
-Correction of mutation,by changing the mutated nucleotide sequence to normal.
-Practically difficult,but in principle can be done by homologous recombination.
3. INHIBITION OF GENE EXPRESSION:
-In case of mutations that have a negative dominant effect, the expression of the mutated gene can be
blocked at the DNA / RNA / protein level.
-Examples: This strategy is useful in Cancers caused by inappropriate expression of a gene.
4. GENE THERAPY TO ACHIEVE PHARMACOLOGICAL EFFECTS:
Examples: 1. Introduction of a gene that makes cancer cells susceptible to anticancer drugs.
2. Introduction of a toxic gene whose expression kills cancer cells.
3. Genes of cytokines can be introduced into cells of immune system to enhance their
potential to kill diseased cells.

5. *ADVANCES IN GENE THERAPY SO FAR:
1. Adenosine Deaminase deficiency:
-First attempt at gene therapy.
2. Severe Combined Immuno Deficiency:
-SCID-X1 successfully treated with gene therapy.
3. Hemophilia A & Hemophilia B:
-Ex vivo method using fibroblasts
-Clinical improvement was present.
4. DMD:
-Successful in mice,but human trials not yet.
5. Cystic Fibrosis:
-In vivo trials with Transmembrane Conductance Regulator CFTR.
*RISKS OF GENE THERAPY:
1.Adverse reactions to the virus or new genes.
2.Activation of proto-oncogene leading to formation of oncogene.
3.Introduction of a mutation to the next generation.
(References: Nelson, IAP Textbook, Genetics for clinicians-Shubha Phadke, Medical Genetics-G.P.Pal.)
-Dr.Padmesh.V

6. *ADVANCES IN GENE THERAPY SO FAR:
1. Adenosine Deaminase deficiency:
-First attempt at gene therapy.
2. Severe Combined Immuno Deficiency:
-SCID-X1 successfully treated with gene therapy.
3. Hemophilia A & Hemophilia B:
-Ex vivo method using fibroblasts
-Clinical improvement was present.
4. DMD:
-Successful in mice,but human trials not yet.
5. Cystic Fibrosis:
-In vivo trials with Transmembrane Conductance Regulator CFTR.
*RISKS OF GENE THERAPY:
1.Adverse reactions to the virus or new genes.
2.Activation of proto-oncogene leading to formation of oncogene.
3.Introduction of a mutation to the next generation.
(References: Nelson, IAP Textbook, Genetics for clinicians-Shubha Phadke, Medical Genetics-G.P.Pal.)
-Dr.Padmesh.V